Photodynamic therapy ("PDT") is an approved cancer treatment that can be used for many purposes, such as the treatment of solid tumors; the impairment of blood-borne targets such as leukemic cells, immunoreactive cells, and unwanted microorganisms; the prevention of restenosis; the treatment of ocular neovascular disorders such as macular degeneration; and the removal of atherosclerotic plaque. PDT involves the topical or systemic application of a light-absorbing photosensitive agent, usually a porphyrin derivative, which accumulates selectively in target tissues. A particularly potent photosensitizer is benzoporphyrin derivative mono-acid ring A ("BPD-MA" or "verteporfin"), which is a second generation chlorin-type photosensitizer exhibiting distinct advances over its hematoporphyrin forerunners in terms of effectiveness at lower concentrations and ability to absorb longer, more penetrating wavelengths of light.
Upon irradiation with visible light of an activating wavelength, reactive oxygen species are produced in cells containing the photosensitizer, which promotes cell death. Evidence has been developed indicating that PDT using a photosensitizer may cause cells to die via an apoptotic pathway. Kessel et al., "Rapid Initiation of Apoptosis by Photodynamic Therapy", Photochem. Photobiol., 63:528-34 (1996); Oleinik et al., "Photodynamic Therapy Induces Rapid Cell Death of Apoptosis in L5178 Mouse Lymphoma Cells", Cancer Res., 51:5993-96 (1991).
Apoptosis is the term used to describe a type of cellular death that occurs in many tissues as a normal physiological process. Apoptosis is a morphologically distinct form of cell death that plays an important role during normal development, differentiation, and homeostasis or turnover of tissues. Also called "programmed cell death," this form of cellular demise involves the activation in cells of a built-in genetic program for cell suicide by which cells essentially autodigest.
The goal of apoptosis is to attain an orderly disintegration of cells into structures suitable for phagocytosis. Morphologically, apoptosis is begun by loss of contact with neighboring cells and smoothening of the cell surface (vesicle formation on the cell surface and membrane "blebbing"). It is further characterized by the concentration of the cytoplasm, endonuclease activity-associated chromatin condensation and pyknosis, and segmentation of the nucleus. The orderly disintegration of cells also includes the degradation of genomic DNA into nucleosomal fragments and cellular fission to form apoptotic bodies. The nucleosome units of the resulting DNA fragments are about 180-200 bases in size. The final fragments of apoptotic body cells are phagocytosed by neighboring cells. The remnants of these dead cells are then cleared almost without a trace by neighboring phagocytic cells, without resulting in inflammation or scarring.
Apoptosis thus stands in marked contrast to necrotic cell death caused, for example by oxygen-deprivation in myocardial infarction or stroke, where cells lose their energy supplies, rupture and spill their contents into the extracellular milieu. Morphologically, necrosis is characterized by marked swelling of mitochondria, swelling of cytoplasm and nuclear alteration, followed by cell destruction and autolysis. It occurs passively or incidentally. Tissue necrosis is generally caused by physical trauma to cells or a chemical poison.
The concept that apoptosis is a finely regulated process is now well established. Kerr et al., "Apoptosis: A Basic Biological Phenomenon with Wide-ranging Implications in Tissue Kinetics", Br. J. Cancer, 26:239-45 (1972). However, the precise molecular mechanism remains as yet uncharacterized.
Apoptosis is thus known to be involved in developmental and tissue specific processes that require the removal of cell populations. In addition to the normal physiological process where cells are turned over within the body, apoptosis can be induced to occur by cellular, hormonal or other stimuli to remove unwanted cells from the body. For example, apoptosis is also known to be involved in the immunological process of cell selection. Specifically, the killing of tumor cells and virus-infected cells by the immune system's cytolytic T-cells occurs via apoptosis following target recognition. Further, apoptosis accounts for cell death in a wide variety of clinically important areas. For example, essentially all chemotherapeutic drugs currently used in the treatment of cancer, as well as x-irradiation in may cases, ultimately kill malignant cells by activating intracellular pathways leading to apoptosis.
Dysregulation of apoptosis, however, may be involved in the pathogenesis of a number of disease states and pathological conditions, such as cancer, acquired immunodeficiency syndrome (AIDS), and neurodegenerative disorders. Specifically, during spontaneous tumor regression, tumor cell death has been shown to follow an apoptotic pathway. During HIV infection, virally induced T-cell death has been shown to follow an apoptotic pathway. And the death of neurons that occurs in diseases such as Alzheimer's dementia and Parkinson's disease shows many hallmarks of apoptosis.
Control of apoptosis has been shown to be useful with respect to specific cells having crucial relevance to developmental biology. Additionally, it would be useful to control apoptosis with respect to treatments involving viral and bacterial pathogens. Cancer chemotherapy could also be enhanced by controlling apoptotic pathways. Efforts have been made using conventional chemotherapy to treat many of the disease states that result in inappropriate apoptotic cell death, but have so far yielded only minor progress toward effective treatment.
The chemical induction of apoptosis is target cell dependent. Glucocorticoids, such as dexamethasone, have been shown to induce apoptosis in thymocytes. Cycloheximide, a known inhibitor of protein synthesis, and actinomycin D, a known inhibitor of mRNA transcription, have also been shown to be powerful inducers of apoptosis in many cell lines. Other inducers of apoptosis include UV irradiation, captothecin, aphidocholin, cisplatin, vincristine, and phorbol myristate acetate plus ionomycin, glucocorticoids, atrophy of hormone-dependent tissues, NK cell, killer cells, tumor necrosis factor (TNF), lymphotoxin (LT), and other cytokines.
The inhibition of apoptosis is also target cell dependent. In addition to being classified as apoptosis inducers, actinomycin D and cycloheximide have also been classified as powerful inhibitors of apoptosis in many cell lines. Other known apoptosis inhibitors include various endonuclease inhibitors, e.g., Zn.sup.2+ and aurintricarboxylic acid.
Inhibition of apoptotic deletion of autoreactive T-cell clones may be achieved by treatment with immunosuppressant cyclosporin A. Other special inhibitors of apoptosis include various steroids and interleukins. The latter stage of apoptosis, i.e., the induction of fission events leading to the formation of apoptosis bodies, may be inhibited by the use of microfilament-disrupting agents, such as cytochalasin B and staurosporin. Agents that inhibit the expression of the oncogene cMyc or that cause the over-expression of the proto-oncogene bcl-2 can inhibit the induction of apoptosis. Calcium ion (Ca.sup.+2) chelating agents; hematopoietic system cytokines, such as IL-3, granulocyte macrophage colony stimulating factor and granulocyte colony stimulating factor; IL-2; and the bcl-2 gene product have all been reported as being capable of repressing apoptosis.
Other apoptosis inhibitors include the carbostyril derivatives of Nakai et al., U.S. Pat. No. 5,464,833 issued Nov. 7, 1995; the unstable dynemicin-like enediyne compounds of Nicolaou, U.S. Pat. No. 5,500,432 issued Mar. 19, 1996; methods of decreasing the activity of the Bcl gene as described by Reed, U.S. Pat. No. 5,550,019 issued Aug. 17, 1996; and compositions containing phytogenic apoptosis inhibitors ("PAls") isolated from plants by Bathurst et al., U.S. Pat. No. 5,567,425 issued Oct. 22, 1996.
In the past few years, it has been shown that the proteolytic cleavage of key cellular substrates represents an important part of the biochemical events underlying apoptosis. Casciola-Rosen et al., "Apopain/CPP32 Cleaves Proteins That Are Essential for Cellular Repair: A Fundamental Principle of Apoptotic Death", J. Exp. Med., 183:1957-64 (1996).
In the past, efforts to identify the cellular components involved in the apoptotic pathway have focused on identifying the signaling molecules and endonucleases capable of cleaving DNA at internucleosomal sites. However, recently, the emphasis has shifted toward examining the role of specific proteases in this process, in particular, the members of the interleukin 1.beta.-converting enzyme ("ICE") family of cysteine proteases. Martin et al., "Protease Activation During Apoptosis: Death by a Thousand Cuts?", Cell., 82:349-52 (1995).
One of the best described pro-apoptotic genes, CED-3, encodes a protein that is highly homologous to the mammalian ICE. Nicholson et al., "Identification and Inhibition of the ICE/CED-3 Protease Necessary for Mammalian Apoptosis", Nature, 376:37-43 (1995). ICE was the first-identified member of a class of cysteine proteases with nearly absolute specificity for aspartic acid residues. Nicholson et al., supra, and Martin et al., supra. The involvement of ICE proteases in both ultraviolet light (UV) and Fas-mediated killing has been well-documented. Casciola-Rosen et al., supra; Muzio et al., "FLICE, a Novel FADD-Homologous ICE/CED-3-like Protease, Is Recruited to the CD95 (Fas/APO-1) Death-inducing Signaling Complex", Cell, 85:817-27 (1996); Yoon et al., "Poly (ADP-ribosyl)ation of Histone HI Correlates with Internucleosomal DNA Fragmentation During Apoptosis", J. Biol. Chem., 271:9129-34 (1996); Chow et al., "Involvement of Multiple Proteases During Fas-mediated Apoptosis in T Lymphocytes", FEBS Lett., 364:134-38 (1995); and Schlegel et al., "CPP32/Apopain is a Key Interleukin 1.beta. Converting Enzyme-like Protease Involved in Fas-mediated Apoptosis", J. Biol. Chem., 271:1841-44 (1996). A number of different homologs in the ICE family have been characterized, as follows:
Homolog Reference Yama/CPP32/Apopain Nicholson et al., supra,; Fernandes- Alnemri et al., "CPP32, A Novel Human Apoptotic Protein with Homology to Caenorhabditis elegans Cell Death Protein CED-3 and Mammalian Interleukin 1.beta.-Converting Enzyme", J. Biol. Chem., 269: 269- 30761-64 (1994); and Salvesen et al., "Yama/CPP32.beta., a Mammalian Homolog of CED-3, Is a CrmA- Inhibitable Protease that Cleaves the Death Substrate Poly(ADP-ribose) Polymerase", Cell., 81: 801-809 (1995). Nedd-2/ICH-1 Kumar et al., "Induction of Apoptosis by the Mouse Nedd2 Gene, Which Encodes a Protein Similar to the Product of the Caenorhabditis elegans Cell Death Gene ced-3 and the Mammalian IL-1-Converting Enzyme", Genes Dev., 8: 1613-26 (1994); and Wang et al., "Ich-1, an ICE/ced-3- related Gene, Encodes Both Positive and Negative Regulators of Programmed Cell Death", Cell., 78: 739-50 (1994). Tx/ICH-2/ICE rel-II Gaucheu et al., "A Novel Human Protease Similar to the Interleukin 1.beta. Converting Enzyme Induces Apoptosis in Transfected Cells", EMBO J., 14: 1914-22 (1995); Kamens et al., "Identification and Characterization of ICH-2, a Novel Member of the Interleukin 1.beta.-Converting Enzyme Family of Cysteine Proteases", J. Biol. Chem., 270: 15250-56 (1995); and Munday, et al., "Molecular Cloning and Proapoptotic Activity of ICE rel-II and ICE rel III, Members of the ICE/CED-3 Family of Cysteine Proteases", J. Biol. Chem., 270: 1587O-76 (1995). ICE rel III Munday et al., supra. Mch-2 Fernandes-Alnemri et al., "Mch-2, a New Member of the Apoptotic CED- 3/ICE Cysteine Protease Gene Family", Cancer Res., 55: 2737-42 (1995). ICE-LAP3/Mch-3/CMH-1 Duan et al., "ICE-LAP3, a Novel Mammalian Homolog of the Caenorhabditis elegans Cell Death Protein CED-3, Is Activated During Fas- and Tumor Necrosis Factor- induced Apoptosis", J. Biol. Chem., 271: 35013-35 (1996); Fernandes- Alnemri, "Mch-3, a Novel Human Apoptotic Cysteine Protease Highly Related to CPP32", Cancer Res., 55: 6045-52 (1995); and Lippke et al., "Identification and Characterization of CPP32/Mch-2 Homolog 1, a Novel Cysteine Protease Similar to CPP32", J. Biol. Chem. 271: 1825-28 (1996). ICE LAP6 Muzio et al., supra.
The ectopic expression of these ICE homologs has been shown to cause apoptosis in a variety of cell types. However, while cysteine proteases of the ICE/CED-3 family have been shown to play an important role in apoptosis induced by various deleterious and physiological stimuli, only CPP32 ("Caspase-3/YAMA/apopain"; see Cell, 87:171 (1996)) and ICE-LAP3 have been shown to be proteolytically activated by conventional apoptotic stimuli. Muzio et al., supra.
The ICE homolog, CPP32, is a cysteine protease that is proteolytically activated by a variety of apoptotic stimuli. Muzio et al., supra. During UV light and Fas-mediated apoptosis, CPP32 is proteolytically cleaved from its precursor 32 kD (p32) to form the active enzyme composed of 17 kD (p17) and 12 kD (p12) subunits. Casciola-Rosen et al., "Apopain/CPP32 Cleaves Proteins That Are Essential for Cellular Repair: A Fundamental Principle of Apoptotic Death", J. Exp. Med., 183:1957-64 (1996); Schlegel et al., "CPP32/Apopain is a Key Interleukin 1.beta. Converting Enzyme-like Protease Involved in Fas-mediated Apoptosis", J. Biol. Chem., 271:1841-44 (1996).
Many of the known proteolytic targets of ICE proteases are proteins associated with the cell nucleus, including lamins (major constituents of the nuclear envelope), globular actin, the nuclear mitotic apparatus protein NuMA, and the U1-70 kD protein (a component of the RNA splicing machinery). Proteolytic targets of ICE proteases also include other enzymes such as poly(ADP-ribose) polymerase (PARP) and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). Askansas et al., "Proteolysis and the Biochemistry of Life-or-death Decisions", J. Exp. Med., 183:1947-51 (1996).
Even though the lamin protease is distinct from the CPP32 protease, cleavage of lamins, in particular, appears to be required for packaging condensed chromatin into apoptotic bodies. Takahashi et al., "Cleavage of Lamin A by Mch2.alpha. But Not CPP32: Multiple Interleukin 1.beta.-converting Enzyme-related Proteases with Distinct Substrate Recognition Properties Are Active in Apoptosis", Proc. Natl. Acad. Sci., 93:8395-8400 (1996); Lazebnik et al., "Studies of the Lamin Proteinase Reveal Multiple Parallel Biochemical Pathways During Apoptotic Execution", Proc. Natl. Acad. Sci., 92:9042-46 (1995).
PARP is an enzyme that appears to serve in the surveillance and enzymatic repair of DNA damage caused by environmental stress. Nicholson et al., "Identification and Inhibition of the ICE/CED-3 Protease Necessary for Mammalian Apoptosis", Nature, 376:37-43 (1995); Kaufmann et al., "Specific Proteolytic Cleavage of Poly(ADP-ribosyl) Polymerase: An Early Marker of Chemotherapy-induced Apoptosis", Cancer Res., 53:3976-85 (1993); Lazebnik et al., "Cleavage of Poly(ADP-ribose) Polymerase by a Proteinase with Properties Like ICE", Nature 371:346-47 (1994). Furthermore, the Ca.sup.++ /Mg.sup.++ -dependent endonuclease that is involved in the internucleosomal cleavage of DNA within apoptotic cells is negatively regulated by poly(ADP-ribos)ylation. Nicholson et al., supra. It has been postulated that loss of normal PARP function may render this nuclease highly active in dying cells. Id.
It has also been demonstrated that CPP32/Yama/Apopain is the protease responsible for the cleavage of PARP. Id.; Fernandes-Alnemri et al., "CPP32, a Novel human Apoptotic Protein with Homology to Caenorhabditis elegans Cell Death Protein CED-3 and Mammalian Interleukin 1.beta.-Converting Enzyme", J. Biol. Chem., 269:30761-64 (1994); and Salvesen et al., "Yama/CPP32.beta., a Mammalian Homolog of CED-3, Is a CrmA-inhibitable Protease that Cleaves the Death Substrate Poly(ADP-ribose) Polymerase", Cell., 81:801-809 (1995). It has been shown that PARP (p116) is cleaved into 85 kD and 25 kD fragments under pro-apoptotic stimuli. Nicholson et al., supra; Salvesen et al., supra; Kaufmann et al., supra; Lazebnik et al., supra; and Enari et al., "Sequential Activation of ICE-like and CPP32-like Proteases During Fas-mediated Apoptosis", Nature, 380:723-26 (1996).
The basic response mechanism of apoptotic cell death that is induced by PDT has been studied, for example, by Jamieson et al., "Efficacy of Benzoporphyrin Derivative, a Photosensitizer, in Selective Destruction of Leukemia Cells Using a Murine Tumor Model", Exp. Haematol., 21:629-34 (1993); Gomer et al., "Molecular, Cellular, and Tissue Response Following Photodynamic Therapy", Las. Surg. Med., 8:450-63 (1988); Gluck et al., "The Selective Uptake of Benzoporphyrin Derivative Mono-acid Ring A Results in Differential Cell Kill of Multiple Myeloma Cells in vitro", Photochem. Photobiol., 63:846-53 (1996); Kessel et al., "Rapid Initiation of Apoptosis by Photodynamic Therapy", Photochem. Photobiol. 63:528-34 (1996); and Oleinik et al., "Photodynamic Therapy Induces Rapid Cell Death by Apoptosis in L5178 Mouse Lymphoma Cells", Cancer Res., 51:5993-96 (1991)). However, the pattern of protease activation in cells treated with PDT has not yet been addressed.